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Biochemistry. 2004 Oct 5;43(39):12585-91.

Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent.

Author information

1
Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche CNRS 8619, Bât. 430, Université Paris-Sud, 91405 Orsay Cedex, France.

Abstract

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.

PMID:
15449948
DOI:
10.1021/bi049049y
[Indexed for MEDLINE]

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